1. Field of the Invention
The present invention relates to an apparatus and method for printing biomolecular droplets on a substrate, and more particularly, to an apparatus and method which uses an electric charge concentration effect to stably and rapidly print biomolecular droplets which have a small volume and diameter on a desired position of a substrate, with narrow intervals between neighboring biomolecular droplets.
2. Description of the Related Art
As a result of the epoch-making development of the Human Genome Project, there is an increasing need for methods of rapidly providing a large amount of genetic information for the diagnosis, treatment and prevention of genetic disorders. Although the Sanger method of analyzing nucleotide sequences has been constantly developed through the development and automation of a polymerase chain reaction (“PCR”) method, in which DNA is duplicated, the Sanger method is complex, time consuming, labor intensive, expensive and requires a substantial amount of expertise. Thus, a large number of genes cannot be analyzed using the Sanger method. As a result, new systems for analyzing nucleotide sequences are continuously being researched. In the last few years, there have been advances in many fields relating to the manufacture and application of biochips.
A biochip, that is, a biological microchip, includes a solid substrate which is made of, for example, silicon, surface-modified glass, polypropylene, or activated polyacrylamide and is combined with biomolecules, such as nucleic acids, proteins or cells, for example, but is not limited thereto. The biochip can be used to analyze gene developing patterns, genetic defects, protein distribution or various kinds of reaction patterns.
If a target material to be analyzed is applied to the biochip, the target material hybridizes with probes immobilized on the biochip. The hybridization is optically or radiochemically detected and analyzed to identify the target material. For example, if a fragment of target DNA to be analyzed is applied to a DNA chip (or DNA microarray) having probes, the target DNA complementarily hybridizes with the probes immobilized on the DNA chip. The hybridization is detected and analyzed using various detecting methods to identify the nucleotide sequence of the target DNA. This is known as sequencing by hybridization (“SBH”).
An example of a printing apparatus for manufacturing a biochip or a DNA microarray is disclosed in Korean Patent Laid-Open Publication No. 2005-0040162. FIG. 1 is a schematic cross-sectional view of a printing apparatus 1 disclosed in the above reference for printing biomolecular droplets on a substrate using an electrohydrodynamic phenomenon. FIG. 2 is a schematic view illustrating an electric field generated when voltage is applied to the printing device 1 illustrated in FIG. 1. Referring to FIGS. 1 and 2, the printing device 1 includes: a first electric field forming electrode 4 which is needle-shaped, formed of a conductive material, is disposed vertically, and comprises an accommodating area 2 in which a biomolecular droplet, such as a nucleic acid (e.g., probe DNA, RNA, PNA and LNA), a protein (e.g., antigen and antibody), an oligopeptide, a eukaryotic cell (e.g., human cell, stem cell, animal cell and vegetable cell), a virus or bacteria is accommodated and a nozzle 3 formed on a bottom end of the accommodating area 2 through which the biomolecular droplet is discharged; a substrate 6 disposed below the first electric field forming electrode 4, and including a target surface 5 onto which a biomolecular droplet 10 discharged from the nozzle 3 of the first electric field forming electrode 4 is deposited; and a second electric field forming electrode 7 made of a conductive material, disposed below the first electric field forming electrode 4, and attached to the substrate 6. In addition, a voltage applying device 9 is connected to and applies a voltage to the first and second electric field forming electrodes 4 and 7 via an electrode lead wire 8.
In the printing device 1, when DC and AC voltages are simultaneously applied to the first and second electric field forming electrodes 4 and 7 by driving the voltage applying unit 9, an electric field is generated between the first and second electric field forming electrodes 4 and 7, as illustrated in FIG. 2. An electric force is created around the biomolecular droplet 10 due to interactions in the electric field generated as described above, the biomolecular droplet 10 having a free surface, and a dielectric constant gradient of the atmosphere. Accordingly, the biomolecular droplet 10 suspended from the nozzle 3 is ejected onto the target surface 5 of the substrate 6.
The printing device 1 can form the electric field between the first electric field forming electrode 4 and the substrate 6 when the substrate 6 is made of a conductive material or the second electric field forming electrode 7 made of a conductive material is attached to the substrate 6. Thus, the electrohydrodynamic effect can be generated to print the biomolecular droplet 10. Accordingly, the substrate 6 should be made of a conductive material or the surface of the substrate 6 should be conductive.
As illustrated in FIG. 2, the electric field may not be uniformly generated between the first electric field forming electrode 4 and the second electric field forming electrode 7. And therefore, the biomolecular droplet 10 may not be ejected onto a desired position of the target surface 5.
Also, when the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is less than a predetermined distance, an electric discharge can be generated. Since the electric discharge may change the biochemical characteristics, size and volume of the biomolecular droplet 10, and the surface structure or characteristics of the substrate 6, the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 should be controlled to prevent the generation of the electric discharge. For example, when the substrate 6 is coated with polymethlymethacrylate (“PMMA”) and the coating thickness is 5 μm, the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is more than 750 μm to prevent the generation of the electric discharge. However, requiring a certain distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 limits design of the device. In addition, if the distance between the first electric field forming electrode 4 and the second electric field forming electrode 7 is too great, it is difficult for the biomolecular droplet 10 to be ejected onto the desired position of the target surface 5.
FIG. 3 is a schematic cross-sectional view of another conventional printing apparatus for printing biomolecular droplets on a substrate using the electrohydrodynamic phenomenon. To eject the biomolecular droplet 10 (FIG. 2) onto the desired position of the target surface 5 (FIG. 1), a ring-shaped ground electrode is introduced as a second electrode to form an electric field only in a ring, as illustrated in FIG. 3 (Electric Field Driven Jetting: An Emerging Approach for Processing Living Cells, Biotechnol. J. 2006, 1, 86-94; Electric Field Driven Jetting: Electrohydrodynamic Jet Processing: An Advanced Electric-Field-Driven Jetting Phenomenon for Processing Living Cells Small. 2006, 2,No. 2, 216-219; Electrohydrodynamic Jetting of Mouse Neuronal Cells, Biochemical Journal, Jan. 4, 2006). Referring to the apparatus of FIG. 3, when biomolecular droplets are ejected out of an electrospray needle corresponding to a first electrode by an electric field formed in only the ring-shaped ground electrode, the biomolecular droplets are ejected only within the ring-shaped electrode and reach a substrate. However, although biomolecular droplets are ejected into only the ring-shaped electrode, in order to prevent electrical discharge the ring-shaped electrode must be separated from the electric spray needle, which prevents ejecting biomolecular droplets onto a desired position of the substrate.
FIG. 4 is a schematic cross-sectional view of a conventional printing apparatus for printing biomolecular droplets on a substrate using an electric charge concentration effect as disclosed in Korean Patent Laid-Open Publication No. 2005-0074496, which solves the problems associated with using the electrohydrodynamic phenomenon as described above. Referring to FIG. 4, in the printing apparatus 100a, when an open circuit type voltage applying unit 60a simultaneously applies a DC voltage and an AC voltage to an electric field forming electrode 20a after a biomolecular droplet is supplied to the apparatus 100a, positive charges migrate into the biomolecular droplet 10a (FIG. 5) suspended from a nozzle 23a and negative charges are induced on a substrate 30a which is electrically grounded. Thus, an electric field is formed between the positive charges and the negative charges as illustrated in FIG. 5. Accordingly, when positive charges migrate into the biomolecular droplet 10a, the negative charges are induced on a portion of the substrate 30a which is disposed opposite to the biomolecular droplet 10a, and a force is generated between the positive charges and the negative charges. In the prior art illustrated in FIGS. 4 and 5, the negative charges are induced below the biomolecular droplet 10a, so that the force is concentrated on the bottom of the biomolecular droplet 10a. The biomolecular droplet 10a suspended from the nozzle 23a is ejected onto a target surface 31a of the substrate 30a due to the force, as illustrated in the middle photo of FIG. 6 and in FIG. 7. Thus, the biomolecular droplet 10a is converted into an approximately hourglass-shaped biomolecular droplet 10a, and a neck (e.g., the thinner portion of an hourglass) is formed in the hourglass-shaped biomolecular droplet 10a. Accordingly, when the hourglass-shaped biomolecular droplet 10a suspended from the nozzle 23a is ejected onto the substrate 30a as illustrated in FIG. 7, the positive charges in the biomolecular droplet 10a are cancelled by the negative charges formed on the substrate 30a, resulting in a reduction in force. That is, the force which pulls the hourglass-shaped biomolecular droplet 10a suspended from the nozzle 23a downward is decreased. In addition, a surface tension A between the hourglass-shaped biomolecular droplet 10 and the substrate 30a, and a surface tension B between the hourglass-shaped biomolecular droplet 10a and the electric field forming electrode 20a act in opposite directions, as illustrated in FIG. 7. Thus, the biomolecular droplet 10a is separated at the neck-shaped portion of the hourglass-shaped biomolecular droplet 10a to become two separate biomolecular droplets. Accordingly, the biomolecular droplets are sequentially deposited on the substrate 30a as illustrated in the last photo of FIG. 6.
The substrate 30a of the apparatus 100a is grounded, thus eliminating or reducing any restriction to its material. In addition, negative charges can be induced on the substrate 30a by the positive charges which have migrated to the bottom portion of the biomolecular droplet 10a, enhancing the amount of positive charges (charge concentration) in the biomolecular droplet 10a in comparison to positive charges generated using the electrohydrodynamic phenomenon described in the prior art. Therefore, the biomolecular droplet 10a can be deposited on a desired position of the target surface 31a of the substrate 30a. In addition, a very high force acts so that the biomolecular droplet 10a can be printed with a smaller size and volume than those of the biomolecular droplet in the prior art. Furthermore, the substrate 30a is grounded, which prevents generation of the detrimental electric discharge associated with the electrohydrodynamic phenomenon as described above. As a result, the distance between the electric field forming electrode 20a and the substrate 30a can be freely adjusted. In summary, using the electric charge concentration effect allows the apparatus 100a to print biomolecular droplets with a small size and volume on a desired position of the substrate 30a. 
However, a method of printing a biomolecular droplet with a small volume is required to manufacture a biochip having a high density using the electric charge concentration effect. In particular, the capability to print a biomolecular droplet having a volume as small as that of about 6 or less cells per biomolecular droplet is required in order to research interactions of some cells (e.g. stem cells). Furthermore, to conduct tissue engineering studies of cells (e.g. stem cells), the cells may need to be printed one by one with very small intervals between neighboring biomolecular droplets. In such studies, biomolecular droplet size is important, and the biomolecular droplet should be rapidly printed to assure that droplets have a homogenous volume.
Referring again to FIG. 4, the apparatus 100a, using the electric charge concentration effect instead of the conventional electrohydrodynamic method, is capable of printing a biomolecular droplet 10a which has a small size and volume at a desired position on the substrate 30a. However, it is still difficult to maintain a homogenous volume of the biomolecular droplet 10a and to control the position on which the biomolecular droplet 10a is deposited. There is also a limit to minimizing the small interval desired between biomolecular droplets according to the size of the biomolecular droplets using the methods described above. In addition, the biomolecular droplet 10a suspended from the nozzle 23a is unstable, which makes printing of the biomolecular droplet 10a time-consuming and slow.